Schizophrenia is a complex disorder lacking an effective treatment option for the pervasive and debilitating cognitive impairments experienced by patients. I have been acutely interested in identifying the underlying circuitry alterations tha contribute to these impairments, so that they may be treated, since the start of my doctoral training. Though my focus has been singular, the approaches and conceptual framework used to study this problem have evolved over my scientific career, and this K01 application represents the next major step in that evolution. This K01 application includes research, clinical, and career development, along with teaching/training opportunities to maximize my ability to successfully transition to independence. My long term career goals are to 1) identify and describe the molecular and cellular alterations that contribute to cortical dysfunction in schizophrenia, 2) determine the possible causes of these observed pathologies using animal models, 3) use these findings to develop pathophysiology-based pharmacological treatments for cognitive impairment in schizophrenia patients, 4) establish an independent research laboratory at a top-tier university to accomplish these goals, and 5) mentor students and postdoctoral fellows to contribute to the next generation of scientists and innovators. My short term career goals include 1) master the research techniques proposed, including laser capture microdissection of single cell populations from human and rodent cortex, electron microscopic mitochondrial analysis of human prefrontal cortical tissue, and shRNA and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) paradigms for rodent studies; 2) master experimental design to determine whether disease findings are a cause, compensation, consequence or confound; and 3) successfully transition as a faculty member from Instructor to Assistant Professor at the University of Pittsburgh. Importantly, completing these short terms goals via this K01 award begins to address long term goals 1 - 3, and puts me on a trajectory to accomplish the remaining long term objectives. The current training plan is also augmented with [[formal courses that address each of the major training goals]], interactive workshops focused on scientific career development, and teaching experiences to further broaden my career skill set. The Department of Psychiatry at the University of Pittsburgh's School of Medicine is an ideal environment in which to accomplish these short and long term goals. This department is a national leader in clinical research, treatment and training. Under the primary mentorship of Dr. David Lewis, Chair of Psychiatry, I will have full access to his laboratory and all of the infrastructure support required for the current application. Working memory is a core cognitive function impaired in schizophrenia that depends upon activation of prefrontal cortex (PFC) circuitry. Accordingly, individuals diagnosed with schizophrenia show reduced PFC activation while performing working memory tasks. This lower PFC activation appears to be an integral part of the disease pathophysiology, and not simply a reflection of poor performance. Thus, the cellular and circuitry alterations that underlie lower PFC neuronal activity in schizophrenia must be determined in order to identify appropriate therapeutic targets. This research proposal focuses on determining which of two discrete possible molecular/physiological disturbances is a likely upstream event leading to PFC impairments in schizophrenia. Supporting neuronal excitation represents the largest energy-consuming activity in the brain, supplied by ATP synthesis in mitochondria via oxidative phosphorylation (OXPHOS). Accumulating evidence indicates that expression of the terminal and rate-limiting OXPHOS enzyme, cytochrome c oxidase (COX), is lower in the PFC of schizophrenia subjects. Thus, the research goal of this K01 application is to determine the underlying mechanism contributing to lower levels of COX in the PFC of schizophrenia subjects. Lower COX could be a consequence of chronic reductions in neuronal excitation that lower ATP demand in the affected neurons (Hypothesis 1), or due to deficient COX expression that impairs metabolic capacity in all neurons (Hypothesis 2). Distinguishing between these alternatives has important implications for identifying appropriate therapeutic targets for cortical dysfunction in schizophrenia, as H1 indicates targeted enhancement of excitation, whereas H2 indicates enhancing COX expression to recover mitochondrial respiration. In order to test which hypothesis is most supported by findings in affected individuals, laser microdissection is used to dissect samples of three distinct neuronal populations and quantitative PCR is used to measure the expression of COX-related transcripts (Aim 1.1), and stereological electron microscopy is used to quantify mitochondrial abundance and morphology (Aim 1.2) in the PFC of schizophrenia and healthy comparison subjects. However, because cause- and-effect relationships cannot be determined using human postmortem tissue, experimental animal models are used in Aims 2 and 3 to directly test the mechanisms of H1 and H2. In Aim 2, DREADD pharmacogenetic technique is used to induce long-term reductions in PFC pyramidal cell excitation, and each measure from Aim 1 is assessed. In Aim 3, viral delivery of shRNA approach is used to impair COX availability in the PFC, and each measure from Aim 1 is assessed. Together, these Aims provide a definitive characterization of the disease phenomenon, and extend to proof-of-concept studies in animal models to provide compelling, convergent and conclusive data regarding which mechanism is operative in the illness.